Placental organic anion transporter

ABSTRACT

A novel organic anion transporter gene participating in organic anion transport in the placenta; and an organic anion transporter which is a polypeptide encoded by the gene. A placental organic anion transporter OAT4, more particularly, a placental organic anion transporter OAT4 having the amino acid sequence represented by SEQ ID NO:2 or an amino acid sequence derived therefrom by deletion, substitution or addition of a part of the amino acids thereof. A nucleic acid (preferably DNA) having a base sequence encoding the placental organic anion transporter OAT4 or a base sequence hybridizable therewith under stringent conditions.

This application is a 371 of PCT/JP00/03878, filed Jun. 15, 2000, whichclaims priority to Japanese Patent Application No. 11-187244, filed Jul.1, 1999.

TECHNICAL FIELD

The present invention relates to gene participating in an organic anion(organic negatively-charged ion) transport and to a polypeptide forwhich the gene codes. More particularly, the present invention relatesto an organic anion transporter OAT4 of a placenta type, to gene codingtherefor, to a probe for detecting the gene and to an antibody which isable to recognize the protein.

BACKGROUND OF THE INVENTION

Kidney and liver play an important role in metabolism and excretion ofxenobiotics and pharmaceuticals. Urinary tubule cells of kidney areepithelial cells having a polarity and contact the blood via abasolateral membrane to carry out transfer of various substances. It hasbeen predicted from the physiological studies up to now that a part ofthe organic ion is incorporated into kidney by a transport carrier(transporter) via a basolateral membrane and also that an organic anionproduced by metabolism in cells is excreted by the transporter.

Since organic anion contains pharmaceuticals and environmental toxins ormany of metabolites thereof, an organic anion transport system has beenwidely known as a xenobiotics excretion system or a pharmaceuticalstransport system as well.

Incorporation of an organic anion by urinary tubule cells has beenstudied by an experimental system using an isolated organ perfusionmethod, an isolated cell membrane vesicle system, etc. However,according to the conventional means, it is difficult to analyze theorganic anion transport system via basolateral membrane in detail andthere has been a demand that the transporter per se is isolated andanalyzed.

Organic anion transport is also carried out in the tissues other thankidney and liver. Placenta is a tissue where material exchange isactively carried out between fetus and the mother's body and thesubstances necessary for living organism including saccharides and aminoacids are efficiently transported to fetus from the mother's body via atransporter.

On the other hand, placenta also plays a role as a tissue barrier forfetus against the external environment. Placenta shows a certain type oflimitation to a free transfer of the xenobiotics ingested by mother'sbody to the fetus and a part of such a function is thought to be by aremoval of the xenobiotics from a fetus circulation by a xenobioticexcretion transporter.

In addition, various metabolic reactions also take place even in thebody of the fetus and, as a result, an organic anion is generated. Dueto an anatomical specificity of the fetus, most of excretion of suchmetabolites is done via placenta. It is rational to conclude that anorganic anion transporter is present in placenta and plays such a role.

As such, it is believed that the transport of xenobiotics (particularly,transport of organic anion) in placenta plays an important role for thegrowth and the genetic toxicity of fetus. However, details of thetransfer therein have been unknown than that in kidney and liver.

The present inventors have isolated and reported on an organic aniontransporter OAT1 (J. Biol. Chem., volume 272, pages 18526-18529, 1997),OAT2 (FEBS Letter, 429, pages 179-182, 1998) and OAT 3 (J. Biol. Chem.,volume 274, pages 13675-13680, 1999) which play a central role inkidney, liver, brain, etc. Patent applications for them have been alsofiled already. OAT1, OAT2 and OAT3 are the transporters which are ableto transport many organic anions having different chemical structuresand they carry out the transport of various anionic pharmaceuticals aswell.

Isolation and identification of OAT1, OAT2 and OAT3 show that organicanion transporters form a family. Members of this family have been knownto be expressed not only in organs such as kidney and liver which play acentral role in external excretion of xenobiotics but also in brainwhich forms a tissue barrier.

From those facts, the present inventors have predicted the presence ofan organic anion transporter in placenta as a functional unit of thetissue barrier and as a route for excretion of metabolites of fetus andhave isolated a novel organic anion transporter existing in placenta.

DISCLOSURE OF THE INVENTION

An object of the present invention is to identify and to provide a novelorganic anion transporter gene participating in an organic aniontransport in placenta and also an organic anion transporter which is apolypeptide for which the gene codes. Other objects are obvious from thefollowing descriptions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an organic anion incorporating activity when OAT4 of thepresent invention is expressed in oocytes of Xenopus laevis.

FIG. 2 shows the result of a dynamic test of transport of A) estronesulfate and B) dehydroepiandrosterone sulfate using the oocytes in whichOAT4 of the present invention is expressed.

FIG. 3 shows the affection by the presence of various cations in thetransport of estrone sulfate using the oocytes in which OAT4 of thepresent invention is expressed.

FIG. 4 shows the result of the transport inhibition test of variousorganic substances in an organic anion transport of OAT4 of the presentinvention.

FIG. 5 shows the result of the transport inhibition test of OAT4 byvarious sulfate conjugates and glucuronic acid conjugates.

FIG. 6 a photographic picture showing the result of the northernblotting analysis of the OAT4 gene of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As mentioned already, the present inventors have isolated three organicanion transporters—OAT1, OAT2 and OAT3. They have a homology in an aminoacid sequence to an extent of around 40% each other. Based upon suchsequences, an EST database (expressed sequence tag database) wasretrieved and a novel cDNA having a homology to OAT 1, 2 and 3 wasidentified. Using this cDNA fragment, a novel clone (OAT4) which has notbeen report yet was identified from a human kidney cDNA library andconfirmed to be a placenta type.

Accordingly, the present invention relates to an organic aniontransporter OAT4 of a placenta type and, more particularly, it relatesto an organic anion transporter OAT4 of a placenta type having an aminoacid sequence of SEQ ID NO:2 of the Sequence Listing or having an aminoacid sequence where a part of the amino acids may be deleted and otheramino acid(s) may be substituted therewith or added thereto.

The organic anion transporter OAT4 of a placenta type according to thepresent invention is an organic anion transporter OAT4 of a placentatype which has an ability of incorporating an organic anion such asestrone sulfate, dehydroepiandrosterone sulfate and/or ochratoxin A.

The present invention also relates to a nucleic acid or, preferably, DNAwhich has a base sequence coding for protein having an amino acidsequence of SEQ ID NO:2 of the Sequence Listing or having an amino acidsequence where a part of the amino acids may be deleted and other aminoacid(s) may be substituted therewith or added thereto or has a basesequence being able to hybridize therewith under a stringent condition.It also relates to gene which codes for the above-mentioned aniontransporter OAT4 of a placenta type of the present invention.

The present invention further relates to a nucleic acid or, preferably,DNA which has a base sequence comprising at least continuous 14 basesor, preferably, at least continuous 20 bases of the DNA having the basesequence shown by SEQ ID NO:1 of the Sequence Listing or a complementarychain thereof The nucleic acid such as the said DNA is useful as a probefor the detection, identification or quantitative determination of thegene coding for the above-mentioned organic anion transporter OAT4 of aplacenta type of the present invention.

The present invention furthermore relates to an antibody which is ableto recognize the above-mentioned organic anion transporter OAT4 of aplacenta type of the present invention.

The organic anion transporter OAT4 of the present invention is atransporter which has a substrate selectivity of a broad range having anability of transporting (incorporating) an organic anion having adifferent chemical structure. The organic anion transporter OAT4 of aplacenta type of the present invention has an ability of incorporatingan organic anion such as estrone sulfate, dehydroepiandrosterone sulfateand/or ochratoxin A.

The present inventors have retrieved a EST database which has beenlaid-open to the public based upon a base sequence information for OAT1,OAT2 and OAT3 which were already isolated by the present inventorswhereupon they have obtained a novel cDNA fragment H12876 having ahomology to OAT1, OAT2 and OAT3. Using a probe where this H12876 waslabeled with ³²p, the present inventors screened a human kidney cDNAlibrary which was constituted already.

As a result, a novel cDNA (hOAT4 cDNA) having an organic aniontransporting activity was obtained. Determination of the base sequenceof the resulting cDNA (OAT4 cDNA) was carried out by an automaticsequencer (manufactured by Applied Biosystems) using a specific primerand found to have a base sequence shown in SEQ ID NO:1 of the SequenceListing.

In order to confirm that the resulting OTA4 has an organic aniontransport activity, cRNA (an RNA complementary to cDNA) was preparedfrom a plasmid containing this cDNA according to a method of Sekine, etal. (Sekine, T., et al., J. Biol. Chem., volume 272, pages 18526-18529,1997) and injected into oocytes of Xenopus laevis and the oocytes weresubjected to an incorporation experiment for various organic anions andorganic cations which were labeled with radioisotopes.

The result is shown in FIG. 1. As shown in FIG. 1, it was found that theoocytes expressing the OAT4 incorporated ³H-estrone sulfate,³H-dehydroepiandrosterone sulfate and ³H-ochratoxin A. On the contrary,no incorporation was noted for ¹⁴C-TEA (tetraethylammonium) which is atypical organic cation.

After that, the above-mentioned oocytes into which OAT4 cRNA wasinjected was used for checking the changes in the amount of estronesulfate, dehydroepiandrosterone sulfate and ochratoxin A incorporated byOAT4 of the present invention at various concentrations whereby aMichaelis-Menten dynamic test for an organic anion transport was carriedout.

The result is shown in FIG. 2. As a result, it was found that the OAT4of the present invention increased the incorporated amount of theorganic anion dependently of the concentration. The Km values of estronesulfate and dehydroepiandrosterone sulfate were found to be 1.01±0.15 μMand 0.63±0.04 μM, respectively.

Dependency of the OAT4 of the present invention on sodium in an organicanion transport was investigated. Incorporation of estrone sulfate byOAT4 was tested in the presence of various extracellular cations. Theresult is shown in FIG. 3. As shown in FIG. 3, an incorporation activityof estrone sulfate via OAT4 was noted when sodium ion, choline ion andlithium ion are present as an extracellular ion and, even when theextracellular sodium is substituted with lithium and choline, there wasno change in the transport of estrone sulfate via OAT4 whereupon it wasclarified that OAT4 is an organic anion transporter which wasindependent upon the extracellular sodium.

Further, in order to investigate the substrate selectivity of OAT4 ofthe present invention, various ionic substances were added to anincorporation experimental system of ³H-estrone sulfate and theinfluence thereby was tested (Inhibition Experiment).

The result is shown in FIG. 4. As a result, various anionic substances(such as probenecid, penicillin G, indomethacin, ibuprofen, diclofenac,furosemide, bumetanide, bromosulfophthalein, cholic acid and taurocholicacid) significantly inhibited the transport of ³H-estrone sulfate byOAT4 but cationic substances such as tetraethylammonium and inorganicsulfates did not show an inhibiting action. From the above result, itwas clarified that OAT4 was a multi-selective organic anion transporter.

Since OAT4 of the present invention showed an incorporation activity forconjugates of the two sulfates, i.e. estrone sulfate anddehydroepiandrosterone sulfate, an inhibiting experiment was carried outto check whether various sulfate conjugates and glucuronate conjugatesshow an interaction with OAT4. The result is shown in FIG. 5. As aresult, all sulfate conjugates except minoxidil sulfate showed aninteraction with OAT4. On the contrary, glucuronate conjugates showedonly a weak interaction with OAT4 except α-naphthyl-β-glucuronide.

Then, a northern blot analysis was carried out to investigate in whichsite of human tissues the OATA4 of the present is expressed.

Result of the northern blotting is shown in FIG. 6. As a result, it wasfound that a strong band was detected in kidney and placenta only in theOAT4 of the present invention and accordingly that the OAT4 of thepresent invention was a placenta type.

Accordingly, the OAT4 of the present invention is an organic aniontransporter of a human placenta type and, in the present invention, thatis called an organic anion transporter OAT4 of a placenta type.

With regard to the protein of the present invention, in addition to thatwhich has an amino acid sequence shown by SEQ ID NO:2, those in whichone or more amino acid(s) in the amino acid sequence shown by SEQ IDNO:2 is/are deleted, substituted or added may be exemplified. Deletion,substitution or addition of amino acid(s) may be within such an extentthat an organic anion transport activity is not lost and the number(s)is/are usually 1 to about 110 or, preferably, from 1 to about 55 aminoacid(s). Such an amino acid has a homology in terms of an amino acidsequence to the amino acid sequence shown by SEQ ID NO:2 to an extent ofusually by 75% or, preferably, by 90%.

In the present invention, hybridization under a stringent condition isusually carried out in such a manner that hybridization is conducted ina hybridization solution of 5×SSC or similar salt concentration theretoat the temperature condition of 37-42° C. for about 12 hours, apreliminary washing is conducted with a solution of 5×SSC or similarsalt concentration thereto and then a washing is conducted with asolution of 1×SSC or similar salt concentration thereto. In order toachieve a higher stringency, it can be done where a washing is conductedin a solution of 0.1×SSC or similar salt concentration thereto.

Nucleic acid which can be subjected to a hybridization in a stringentcondition in the present invention includes that which can be subjectedto a hybridization under the above-mentioned conditions.

It is also possible that the organic anion transporter and gene thereofaccording to the present invention is isolated and obtained by means ofa screening where tissues or cells of appropriate mammal besides humanbeing are used as a gene source. With regard to the mammal, non-humananimals such as dogs, cattle, horses, goats, sheep, monkeys, pigs,rabbits, rats and mice may be used. Screening and isolation of the genemay be advantageously carried out by means of a homology screening, aPCR screening, etc.

As to the resulting cDNA, its base sequence is determined by aconventional method and a translated region is analyzed whereby theprotein coded therefor or the amino acid sequence of OAT4 can bedetermined.

The fact that the resulting cDNA is a cDNA of an organic aniontransporter or, in other words, the gene product coded for cDNA is anorganic anion transporter can, for example, be inspected as follows.Thus, cRNA prepared from the resulting OAT4 cDNA is introduced intooocytes to express and an ability of transporting (incorporating) theorganic anion into cells can be confirmed by measuring the expression ofthe substrate into the cells by means of a conventional expressionexperiment (Sekine, et al., J. Biol. Chem., volume 272, pages18526-18529, 1997) using an appropriate anion as a substrate.

It is also possible that the expression experiment which is as same asin the above-mentioned expressed cells is applied to investigate thetransport characteristic and the substrate specificity of OAT4.

When an appropriate cDNA library or genomic DNA library prepared from adifferent genetic source is screened using the OAT4 cDNA, it is possibleto isolate homologous gene, chromosomal gene, etc. derived fromdifferent tissues and different living bodies.

It is further possible to isolate the gene from a cDNA library by aconventional PCR method using a synthetic primer designed based on theinformation of the disclosed base sequence (or a part of it) shown inthe base sequence (SEQ ID NO:1) of the gene of the present invention.

The DNA library such as a cDNA library and a genomic DNA library can beprepared by a method mentioned, for example, in Molecular Cloning bySambrook, J., Fritsh, E. F. and Maniatis, T., published by Cold SpringHarbor Laboratory Press, 1989. When a library is commercially available,that may be used as well.

The organic anion transporter (OAT4) of the present invention may beproduced, for example, by means of a gene recombination technique usingcDNA coding for the organic anion transporter. For example, DNA (cDNA orthe like) coding for the organic anion transporter is incorporated intoan appropriate expression vector and the resulting recombinant DNA canbe introduced into an appropriate host cell. Examples of the expressionsystem (host vector system) for the production of a polypeptide areexpression systems of bacteria, yeast, insect cells and mammalian cells.In order to obtain a functional protein among them, it is desirable touse insect cells and mammalian cells.

For example, in order to express a polypeptide in mammal, DNA coding foran organic anion transporter is inserted into a downstream to anappropriate promoter (such as SV40 promoter, LTR promoter and elongation1α promoter) in an appropriate expression vector (such as vector of aretrovirus, papilloma virus, vaccinia virus vector and vector of an SV40type) whereupon an expression vector is constructed. Then appropriateanimal cells are subjected to a transformation using the resultingexpression vector and the transformant is incubated in an appropriatemedium to give a desired polypeptide. Examples of the mammal cells usedas a host are cell strains including simian COS-7 cells, Chinese hamsterCHO cells, human Hela cells, primary culture cells derived from kidneytissues, LLC-PK1 cells derived from kidney of pig, OK cells derived fromopossum kidney, etc.

With regard to cDNA which codes for an organic anion transporter OAT4,the cDNA having a base sequence shown in SEQ ID NO: 1 may be used forexample and, in addition, it is not limited to the above-mentioned cDNAbut DNA corresponding to the amino acid sequence is designed and the DNAcoding for the polypeptide may be used. In that case, 1-6 kind(s) ofcodon has/have been known for coding for each amino acid and, althoughany codon may be selected for use, a sequence having higher expressioncan be designed when, for example, frequent use of codon by the hostutilized in the expression is taken into consideration. Like in the caseof chemical synthesis of DNA and fragmentation of the above cDNA, theDNA having a designed base sequence can be prepared, for example, bymeans of a partial modification of the base sequence. Artificial partialmodification of and introduction of variation into a base sequence canbe carried out by a site-directed mutagenesis (Mark, D. F., et al: Proc.Natl. Acad. Sci. USA, volume 18, pages 5662-5666, 1984), etc. utilizinga primer comprising a synthetic oligonucleotide coding for the desiredmodification.

The nucleotide (oligonucleotide or polynucleotide) which hybridizes tothe organic anion transporter gene of the present invention under astringent condition can be used as a probe for detecting the organicanion transporter gene and, moreover, it can be used as antisenseoligonucleotide, ribozyme, decoy, etc. for modulation of expression ofthe organic anion transporter. With regard to such a nucleotide, anucleotide containing a partial sequence of usually not less thancontinuous 14 bases in the base sequence shown by SEQ ID NO:1 or acomplementary sequence thereof may be used for example and, in order tohybridize more specifically, longer sequence such as not less than 20bases or not less than 30 bases may be used as a partial sequence.

It is also possible that the organic anion transporter of the presentinvention or a polypeptide having the immunological homology thereto isused to obtain an antibody thereto and the antibody is able to beutilized for detection, purification, etc. of the organic aniontransporter. The antibody can be manufactured using the organic aniontransporter of the present invention or a fragment thereof or asynthetic peptide having a partial sequence thereof as an antigen.Polyclonal antibody can be manufactured by a conventional method inwhich an antigen is inoculated to a host animal (such as rat and rabbit)and immune serum is obtained while monoclonal antibody can bemanufactured by a conventional way such as a hybridoma method.

EXAMPLES

The present invention will now be further illustrated by way of thefollowing Examples although those Examples do not limit the presentinvention.

In the following Examples, each operation is carried out, unlessotherwise mentioned, by a method mentioned in Molecular Cloning bySambrook, J., Fritsh, E. F. and Maniatis, T., published by Cold SpringHarbor Laboratory Press, 1989 or according to the direction for use ofthe commercially available product when commercially available kit isused.

Example 1 Isolation of Multi-Specific Organic Anion Transporter 4 (OAT4)cDNA and Analysis Thereof.

An laid-open EST database was investigated based upon a base sequenceinformation of OAT1, OAT2 and OAT3 which were isolated by the presentinventors already. As a result, a novel cDNA fragment 12876 having ahomology with OAT1, OAT2 and OAT3 was obtained.

A human kidney cDNA library which was constructed already was screenedusing a probe where the resulting H12876 was labeled with ³²p. Ahybridization was carried out for one night and day in a solution forhybridization at 37° C. and, after that, the filter membrane was washedwith a 0.1×SSC/0.1% SDS at 37° C. As to the hybridization solution, abuffer of pH 6.5 containing 50% formamide, 5×standard saline citrate(SSC), 3×Denhardt's solution, 0.2% SDS, 10% dextran sulfate, 0.2 mg/mlmodified salmon semen DNA, 2.5 mM sodium pyrophosphate, 25 mM MES and0.01% Antifoam B (manufactured by Sigma) was used.

Clone which was isolated into λ Zip Lox was further subjected to asubcloning to a plasmid vector pZL by an in vivo excision method. As aresult, a novel cDNA (hOAT4 cDNA) having an organic anion transportingactivity was prepared.

Determination of a base sequence of the above-prepared cDNA (OAT4 cDNA)was carried out by an automatic sequencer (manufactured by AppliedBiosystems) using a specific primer. The base sequence is shown in SEQID NO:1 of the Sequence Listing.

Example 2 Characterization of OAT4 Function.

From an OAT4 cDNA-containing plasmid was prepared cRNA (an RNAcomplementary to cDNA) in vitro using a T7 RNA polymerase (Sekine, T.,et al., J. Biol. Chem., volume 272, pages 18526-18529, 1997).

The resulting cRNA was injected into oocytes of Xenopus laevis accordingto the already-reported method (Sekine, T., et al., J Biol. Chem.,volume 272, pages 18526-18529, 1997) and the oocytes were subjected toan incorporation experiment using various labeled organic anions andorganic cations. As a result, it was found as shown in FIG. 1 that theoocytes in which OAT4 was expressed showed incorporation of ³H-estronesulfate, ³H-dehydroepiandrosterone sulfate and ³H-ochratoxin A. On thecontrary, uptake of ¹⁴C-TEA (tetraethylammonium) which is a typicalorganic cation was not noted.

Example 3 Kinetic Test of OAT4 for Organic Anion Transport.

A Michaelis-Menten dynamic test was carried out for an organic aniontransport of OAT4. Changes in the uptake amount of variousconcentrations of estrone sulfate and dehydroepiandrosterone sulfate byOAT4 were tested whereby concentration dependency of those substrates byOAT4 was investigated. The uptake experiment of labeled estrone sulfateand dehydroepiandrosterone sulfate was carried out according to theabove-mentioned method using oocytes into which OAT4 cRNA was injected.

As a result, the Km values of estrone sulfate and dehydroepiandrosteronesulfate were 1.01±0.15 μM and 0.63±0.04 μM, respectively (refer to FIG.2).

Example 4 Test on Cation-Dependency in Organic Anion Transport of OAT4.

Sodium-dependency of OAT4 in an organic anion transport wasinvestigated. Even when extracellular sodium was substituted withlithium and choline, there was no change in the transport of estronesulfate via OAT4 and it was clarified that OAT4 is an organic aniontransporter which is independent on extracellular sodium (refer to FIG.3).

In order to further investigate the substrate selectivity of OAT4,various anionic substances were added to a system in an uptakeexperiment system of ³H-estrone sulfate in oocytes into which OAT4 cRNAwas injected and the influence was investigated (inhibition experiment).An uptake experiment of ³H-estrone sulfate was carried out according tothe above-mentioned method using oocytes into which OAT4 cRNA wasinjected. Uptake of 50 nM ³H-estrone sulfate was measured in thepresence and absence of 500 μM of various compounds (unlabeled). Theresult was that various anionic substances (such as probenecid,penicillin G, indomethacin, ibuprofen, diclofenac, furosemide,bumetanide, bromosulfophthalein, cholic acid and taurocholic acid)significantly inhibited the transport of ³H-estrone sulfate by OAT4(refer to FIG. 3). On the other hand, cationic substances such astetraethylammonium and inorganic sulfates did not show any inhibitingaction (refer to FIG. 4). From the above results, it was clarified thatOAT4 is a multi-specific organic anion transporter.

Example 5 Uptake Test of Various Sulfate Conjugates and Glucuronic AcidConjugates by OAT4.

OAT4 showed an uptake activity for conjugates of the two sulfate—estronesulfate and dehydroepiandrosterone sulfate—and, therefore, an inhibitorytest was carried out to see whether various sulfate conjugates andglucuronic acid conjugates also show an interaction with OAT4. As shownin FIG. 5, all sulfate conjugates except minoxidil sulfate showed aninteraction with OAT4. On the contrary, glucuronic acid conjugatesshowed only weak interaction with OAT4 except α-naphthyl-β-glucuronide.

Example 6 Northern Blotting Analysis of OAT4 Gene.

Analysis of expression (northern blotting) of OAT4 gene in varioustissues of human being was carried out. Full length of OAT4 cDNA waslabeled with ³²p-dCTP and this was used as a probe for carrying out ahybridization of a filter (manufactured by Clonetec) where RNA extractedfrom various tissues of human being was blotted. Hybridization wascarried out overnight using a hybridization solution containing the fulllength of OAT4 cDNA and the filter was washed at 65° C. with a 0.1×SSCcontaining 0.1% SDS. As a result of northern blotting, a strong band wasdetected only in the cases of kidney and placenta (refer to FIG. 6).Incidentally, the blots in FIG. 6 shows brain, heart, skeletal muscle,colon, thymus, spleen, kidney, liver, small intestine, placenta, lungand peripheral blood leukocytes from left to right.

Industrial Applicability

The present invention provides a novel organic anion transporter OAT4 ofa placenta type and also a gene coding therefor. A transporter is aprotein which incorporates or excretes a substance necessary for themaintenance of life of cells as same as a channel and its abnormalitycauses various diseases. Particularly, the organic anion transporterOAT4 of a placenta type according to the present invention isselectively expressed in kidney and placenta and its clarification isuseful for prevention and treatment of various renal diseases andabnormal growth of fetus.

2 1 2210 DNA Human 1 gttccaaaca gcagttaggt cagcagtccg ctcagccgaggcagctctgt tcatggcgtt 60 ctcgaagctc ttggagcaag ccggaggcgt gggcctcttccagaccctgc aggtgctcac 120 cttcatcctc ccctgcctca tgataccttc ccagatgctcctggagaact tctcagccgc 180 catcccaggc caccgatgct ggacacacat gctggacaatggctctgcgg tttccacaaa 240 catgaccccc aaggcccttc tgaccatctc catcccgccaggccccaacc aggggcccca 300 ccagtgccgc cgcttccgcc agccacagtg gcagctcttggaccccaatg ccacggccac 360 cagctggagc gaagctgaca cggagccgtg tgtggacggctgggtctatg accgcagcgt 420 cttcacctcc accatcgtgg ccaagtggga cctggtgtgcagctcccagg gcttgaagcc 480 cctaagccag tccatcttca tgtccgggat cctggtgggctcctttatct ggggcctcct 540 ctcctaccgg tttgggagga agccgatgct gagctggtgctgcctgcagt tggccgtggc 600 gggcaccagc accatcttcg ccccaacatt cgtcatctactgcggcctgc ggttcgtggc 660 cgcttttggg atggccggca tctttctgag ttcactgacactgatggtgg agtggaccac 720 gaccagcagg agggcggtca ccatgacggt ggtgggatgtgccttcagcg caggccaggc 780 ggcgctgggc ggcctggcct ttgccctgcg ggactggaggactctccagc tggcagcatc 840 agtgcccttc tttgccatct ccctgatatc ctggtggctgccagaatccg cccggtggct 900 gattattaag ggcaaaccag accaagcact tcaggagctcagaaaggtgg ccaggataaa 960 tggccacaag gaggccaaga acctgaccat agaggtgctgatgtccagcg tgaaggagga 1020 ggtggcctct gcaaaggagc cgcggtcggt gctggacctgttctgcgtgc ccgtgctccg 1080 ctggaggagc tgcgccatgc tggtggtgaa tttctctctattgatctcct actatgggct 1140 ggtcttcgac ctgcagagcc tgggccgtga catcttcctcctccaggccc tcttcggggc 1200 cgtggacttc ctgggccggg ccaccactgc cctcttgctcagtttccttg gccgccgcac 1260 catccaggcg ggttcccagg ccatggccgg cctcgccattctagccaaca tgctggtgcc 1320 gcaagatttgc agaccctgc gtgtggtctt tgctgtgctgggaaagggat gttttgggat 1380 aagcctaacct gcctcacca tctacaaggc tgaactctttccaacgccag tgcggatgac 1440 agcagatggca ttctgcata cagtgggccg gctgggggctatgatgggtc ccctgatcct 1500 gatgagccgcc aagccctgc ccctgctgcc tcctctcctctatggcgtta tctccattgc 1560 ttccagcctgg ttgtgctgt tcttcctccc ggagacccagggacttccgc tccctgacac 1620 tatccaggacc tggagagcc agaaatcaac agcagcccagggcaaccggc aagaggccgt 1680 cactgtggaaa gtacctcgc tctagaaatt gtgcctgcatggagcccctt tagtcaaaga 1740 ctcctggaaa ggagttgcct cttctccaat cagagcgtggaggcgagttg ggcgacttca 1800 agggcctggc atggcagagg ccaggcagcc gtggccgagtggacagcgtg gccgtctgct 1860 gtggctgaag gcagcttcca cagctcactc ctcttctccctgccctgatc agattcccca 1920 ccttacccgg gccctacagg agcctgtgca gatggccatgcccaaccaat aacgagacgg 1980 ttcccctccc tttccctgcc aggctcatgt ctttacaccttcactcagcc acgccaacca 2040 gagactgggt tccaatctca ccccaccaca tacagagccctcatctgtga aatgagaatg 2100 atcacgtgac ccacccccca gggcaggtat cagggtgaactgatcttagc accggccaaa 2160 taaatggaac ctgctgagag agctgccaga taaaaaaaaaaaaaaaaaaa 2210 2 550 PRT Human 2 Met Ala Phe Ser Lys Leu Leu Glu GlnAla Gly Gly Val Gly Leu Phe 1 5 10 15 Gln Thr Leu Gln Val Leu Thr PheIle Leu Pro Cys Leu Met Ile Pro 20 25 30 Ser Gln Met Leu Leu Glu Asn PheSer Ala Ala Ile Pro Gly His Arg 35 40 45 Cys Trp Thr His Met Leu Asp AsnGly Ser Ala Val Ser Thr Asn Met 50 55 60 Thr Pro Lys Ala Leu Leu Thr IleSer Ile Pro Pro Gly Pro Asn Gln 65 70 75 80 Gly Pro His Gln Cys Arg ArgPhe Arg Gln Pro Gln Trp Gln Leu Leu 85 90 95 Asp Pro Asn Ala Thr Ala ThrSer Trp Ser Glu Ala Asp Thr Glu Pro 100 105 110 Cys Val Asp Gly Trp ValTyr Asp Arg Ser Val Phe Thr Ser Thr Ile 115 120 125 Val Ala Lys Trp AspLeu Val Cys Ser Ser Gln Gly Leu Lys Pro Leu 130 135 140 Ser Gln Ser IlePhe Met Ser Gly Ile Leu Val Gly Ser Phe Ile Trp 145 150 155 160 Gly LeuLeu Ser Tyr Arg Phe Gly Arg Lys Pro Met Leu Ser Trp Cys 165 170 175 CysLeu Gln Leu Ala Val Ala Gly Thr Ser Thr Ile Phe Ala Pro Thr 180 185 190Phe Val Ile Tyr Cys Gly Leu Arg Phe Val Ala Ala Phe Gly Met Ala 195 200205 Gly Ile Phe Leu Ser Ser Leu Thr Leu Met Val Glu Trp Thr Thr Thr 210215 220 Ser Arg Arg Ala Val Thr Met Thr Val Val Gly Cys Ala Phe Ser Ala225 230 235 240 Gly Gln Ala Ala Leu Gly Gly Leu Ala Phe Ala Leu Arg AspTrp Arg 245 250 255 Thr Leu Gln Leu Ala Ala Ser Val Pro Phe Phe Ala IleSer Leu Ile 260 265 270 Ser Trp Trp Leu Pro Glu Ser Ala Arg Trp Leu IleIle Lys Gly Lys 275 280 285 Pro Asp Gln Ala Leu Gln Glu Leu Arg Lys ValAla Arg Ile Asn Gly 290 295 300 His Lys Glu Ala Lys Asn Leu Thr Ile GluVal Leu Met Ser Ser Val 305 310 315 320 Lys Glu Glu Val Ala Ser Ala LysGlu Pro Arg Ser Val Leu Asp Leu 325 330 335 Phe Cys Val Pro Val Leu ArgTrp Arg Ser Cys Ala Met Leu Val Val 340 345 350 Asn Phe Ser Leu Leu IleSer Tyr Tyr Gly Leu Val Phe Asp Leu Gln 355 360 365 Ser Leu Gly Arg AspIle Phe Leu Leu Gln Ala Leu Phe Gly Ala Val 370 375 380 Asp Phe Leu GlyArg Ala Thr Thr Ala Leu Leu Leu Ser Phe Leu Gly 385 390 395 400 Arg ArgThr Ile Gln Ala Gly Ser Gln Ala Met Ala Gly Leu Ala Ile 405 410 415 LeuAla Asn Met Leu Val Pro Gln Asp Leu Gln Thr Leu Arg Val Val 420 425 430Phe Ala Val Leu Gly Lys Gly Cys Phe Gly Ile Ser Leu Thr Cys Leu 435 440445 Thr Ile Tyr Lys Ala Glu Leu Phe Pro Thr Pro Val Arg Met Thr Ala 450455 460 Asp Gly Ile Leu His Thr Val Gly Arg Leu Gly Ala Met Met Gly Pro465 470 475 480 Leu Ile Leu Met Ser Arg Gln Ala Leu Pro Leu Leu Pro ProLeu Leu 485 490 495 Tyr Gly Val Ile Ser Ile Ala Ser Ser Leu Val Val LeuPhe Phe Leu 500 505 510 Pro Glu Thr Gln Gly Leu Pro Leu Pro Asp Thr IleGln Asp Leu Glu 515 520 525 Ser Gln Lys Ser Thr Ala Ala Gln Gly Asn ArgGln Glu Ala Val Thr 530 535 540 Val Glu Ser Thr Ser Leu * 545 550

What is claimed is:
 1. The isolated organic anion transporter OAT4 of aplacenta type, wherein it has an amino acid sequence of SEQ ID NO: 2 ofthe Sequence listing.
 2. The isolated organic anion transporter OAT4 ofa placenta type according to claim 1 wherein it has an ability oftransporting estrone sulfate, dehydroepiandrosterone sulfate and/orochratoxin A.